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1.  NTHi Induction of Cxcl2 and Middle Ear Mucosal Metaplasia in Mice 
The Laryngoscope  2013;123(11):10.1002/lary.24097.
Objective
Chronic Otitis Media (COM) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute OM pathogen, is known to activate inflammation and mucin expression in vitro and in animal models of OM. The goals of this study were to examine histopathological and expression profiling epithelial effects of NTHi challenge in murine middle ears.
Study Design
In vitro and in vivo murine model of OM.
Methods
Weekly transtympanic inoculation of Balb/c mice with 300 μg/ml of NTHi lysates vs saline was performed. Histopathologic analysis was carried out at 4 weeks. Expression microarray analysis was performed at 1 and 7 days. Microarray findings were validated in independent animal samples and in a cultured murine middle ear epithelial cell (mMEEC) line.
Results
Histopathologic analyses revealed middle ear mucosal thickening after NTHi exposure. Microarray analyses of inflammatory response genes that changed significantly demonstrated that the chemokine Cxcl2 had the largest fold-change with significantly increased expression at 1 and 7 days after NTHi injection compared to either saline or no-injection (p<0.01). Validation by realtime qPCR revealed similar significantly increased relative mRNA levels for Cxcl2. NTHi lysates were also found to significantly up-regulate the transcription of Cxcl2 in mMEEC in a time and dose dependent manner (p<0.05).
Conclusions
Middle ear NTHi challenge in mice leads to chronic epithelial mucosal metaplasia and over-expression of inflammatory mediators, most notably Cxcl2. This finding is parallel to NTHi mediated pulmonary mucosal metaplasia where Cxcl2 has been identified as an important inflammatory mediator.
doi:10.1002/lary.24097
PMCID: PMC3740030  PMID: 23553435
NTHi; Otitis Media; Cxcl2; metaplasia
2.  Mathematical Modeling of Corticosteroid Pharmacogenomics in Rat Muscle following Acute and Chronic Methylprednisolone Dosing 
Molecular pharmaceutics  2008;5(2):328-339.
The pharmacogenomic effects of a corticosteroid (CS) were assessed in rat skeletal muscle using microarrays. Adrenalectomized (ADX) rats were treated with methylprednisolone (MPL) by either 50 mg/kg intravenous injection or 7-day 0.3 mg/kg/h infusion through subcutaneously implanted pumps. RNAs extracted from individual rat muscles were hybridized to Affymetrix Rat Genome Genechips. Data mining yielded 653 and 2316 CS-responsive probe sets following MPL bolus and infusion treatments. Of these, 196 genes were controlled by MPL under both dosing conditions. Cluster analysis revealed that 124 probe sets exhibited three typical expression dynamic profiles following acute dosing. Cluster A consisted of up-regulated probe sets which were grouped into five subclusters each exhibiting unique temporal patterns during the infusion. Cluster B comprised down-regulated probe sets which were divided into two subclusters with distinct dynamics during the infusion. Cluster C probe sets exhibited delayed down-regulation under both bolus and infusion conditions. Among those, 104 probe sets were further grouped into subclusters based on their profiles following chronic MPL dosing. Several mathematical models were proposed and adequately captured the temporal patterns for each subcluster. Multiple types of dosing regimens are needed to resolve common determinants of gene regulation as chronic exposure results in unexpected differences in gene expression compared to acute dosing. Pharmacokinetic/pharmacodynamic (PK/PD) modeling provides a quantitative tool for elucidating the complexities of CS pharmacogenomics in skeletal muscle.
doi:10.1021/mp700094s
PMCID: PMC4196382  PMID: 18271548
Microarray studies; pharmacokinetics; pharmacodynamics; mathematical models; computational biology
3.  Microarray analysis of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of two dosing regimens 
Physiological genomics  2007;30(3):282-299.
The transcriptional response of skeletal muscle to chronic corticosteroid exposure was examined over 168 h and compared with the response profiles observed following a single dose of corticosteroid. Male adrenalectomized Wistar rats were given a constant-rate infusion of 0.3 mg•kg−1•h−1 methylprednisolone for up to 7 days via subcutaneously implanted minipumps. Four control and forty drug-treated animals were killed at ten different time points during infusion. Liver total RNAs were hybridized to 44 individual Affymetrix REA230A gene chips. Previously, we described a filtration approach for identifying genes of interest in microarray data sets developed from tissues of rats treated with methylprednisolone (MPL) following acute dosing. Here, a similar approach involving a series of three filters was applied sequentially to identify genes of interest. These filters were designed to eliminate probe sets that were not expressed in the tissue, not regulated by the drug, or did not meet defined quality control standards. Filtering eliminated 86% of probe sets, leaving a remainder of 2,316 for further consideration. In a previous study, 653 probe sets were identified as MPL regulated following administration of a single (acute) dose of the drug. Comparison of the two data sets yielded 196 genes identified as regulated by MPL in both dosing regimens. Because of receptor downregulation, it was predicted that genes regulated by receptor-glucocorticoid response element interactions would exhibit tolerance in chronic profiles. However, many genes did not exhibit steroid tolerance, indicating that present perspectives on the mechanism of glucocorticoid action cannot entirely explain all temporal profiles.
doi:10.1152/physiolgenomics.00242.2006
PMCID: PMC4186702  PMID: 17473217
glucocorticoids; corticosteroids; Affymetrix gene chips; gene expression; time series
4.  Application of Scaling Factors in Simultaneous Modeling of Microarray Data from Diverse Chips 
Pharmaceutical research  2007;24(4):643-649.
Purpose
Microarrays have been utilized in many biological, physiological and pharmacological studies as a high-throughput genomic technique. Several generations of Affymetrix GeneChip® microarrays are widely used in gene expression studies. However, differences in intensities of signals for different probe sets that represent the same gene on various types of Affymetrix chips make comparison of datasets complicated.
Materials and Methods
A power coefficient scaling factor was applied in the pharmacokinetic/ pharmacodynamic (PK/PD) modeling to account for differences in probe set sensitivities (i.e., signal intensities). Microarray data from muscle and liver following methylprednisolone 50 mg/kg i.v. bolus and 0.3 mg/kg/h infusion regimens were taken as an exemplar.
Results
The scaling factor applied to the pharmacodynamic output function was used to solve the problem of intensity differences between probe sets. This approach yielded consistent pharmacodynamic parameters for the applied models.
Conclusions
Modeling of pharmacodynamic/pharmacogenomic (PD/PG) data from diverse chips should be performed with caution due to differential probe set intensities. In such circumstances, a power scaling factor can be applied in the modeling.
doi:10.1007/s11095-006-9215-y
PMCID: PMC4181592  PMID: 17318415
bioinformatics; computational biology; pharmacodynamics; pharmacogenomics; pharmacokinetics
5.  Differential Gene Expression Reveals Mitochondrial Dysfunction in an Imprinting Center Deletion Mouse Model of Prader-Willi Syndrome 
Clinical and translational science  2013;6(5):10.1111/cts.12083.
Prader-Willi syndrome (PWS) is a genetic disorder caused by deficiency of imprinted gene expression from the paternal chromosome 15q11-15q13 and clinically characterized by neonatal hypotonia, short stature, cognitive impairment, hypogonadism, hyperphagia, morbid obesity and diabetes. Previous clinical studies suggest that a defect in energy metabolism may be involved in the pathogenesis of PWS. We focused our attention on the genes associated with energy metabolism and found that there were 95 and 66 mitochondrial genes differentially expressed in PWS muscle and brain, respectively. Assessment of enzyme activities of mitochondrial oxidative phosphorylation (OXPHOS) complexes in the brain, heart, liver and muscle were assessed. We found the enzyme activities of the cardiac mitochondrial complexes II+III were upregulated in the imprinting center deletion (PWS-IC) mice compared to the wild type littermates. These studies suggest that differential gene expression, especially of the mitochondrial genes may contribute to the pathophysiology of PWS.
doi:10.1111/cts.12083
PMCID: PMC3815468  PMID: 24127921
6.  An Exploration of Heat Tolerance in Mice Utilizing mRNA and microRNA Expression Analysis 
PLoS ONE  2013;8(8):e72258.
Background
Individuals who rapidly develop hyperthermia during heat exposure (heat-intolerant) are vulnerable to heat associated illness and injury. We recently reported that heat intolerant mice exhibit complex alterations in stress proteins in response to heat exposure. In the present study, we further explored the role of genes and molecular networks associated with heat tolerance in mice.
Methodology
Heat-induced physiological and biochemical changes were assessed to determine heat tolerance levels in mice. We performed RNA and microRNA expression profiling on mouse gastrocnemius muscle tissue samples to determine novel biological pathways associated with heat tolerance.
Principal Findings
Mice (n = 18) were assigned to heat-tolerant (TOL) and heat-intolerant (INT) groups based on peak core temperatures during heat exposures. This was followed by biochemical assessments (Hsp40, Hsp72, Hsp90 and Hsf1 protein levels). Microarray analysis identified a total of 3,081 mRNA transcripts that were significantly misregulated in INT compared to TOL mice (p<0.05). Among them, Hspa1a, Dnajb1 and Hspb7 were differentially expressed by more than two-fold under these conditions. Furthermore, we identified 61 distinct microRNA (miRNA) sequences significantly associated with TOL compared to INT mice; eight miRNAs corresponded to target sites in seven genes identified as being associated with heat tolerance pathways (Hspa1a, Dnajb1, Dnajb4, Dnajb6, Hspa2, Hspb3 and Hspb7).
Conclusions
The combination of mRNA and miRNA data from the skeletal muscle of adult mice following heat stress provides new insights into the pathophysiology of thermoregulatory disturbances of heat intolerance.
doi:10.1371/journal.pone.0072258
PMCID: PMC3744453  PMID: 23967293
7.  Global gene profiling of VCP-associated inclusion body myopathy 
Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder caused by mutations in the Valosin Containing Protein (VCP) gene on chromosome 9p12-13. Patients demonstrate limb girdle muscle weakness, which eventually progresses to involve respiratory muscles, and death from respiratory and cardiac failure. This is the first investigation to analyze key molecular mediators and signaling cascades in skeletal muscle causing myopathy by global gene microarray in hopes of understanding the dysregulated genes and molecular mechanisms underlying IBMPFD and the hope of finding novel therapeutic targets. We determined expression profiles using Human Genome Array microarray technology in Vastus lateralis muscles from patients and their first degree relatives. We analyzed gene annotations by DAVID and identified differentially dysregulated genes with roles in several novel biological pathways, including regulation of actin cytoskeleton, ErbB signaling, cancer, in addition to regulation of autophagy, and lysosomal signaling, known disrupted pathways in VCP disease. In this report, we present data from the first global microarray analyzing IBMPFD patient muscles and elucidating dysregulated pathways to further understand the pathogenesis of the disease and discover potential therapeutics.
doi:10.1111/j.1752-8062.2012.00407.x
PMCID: PMC3375869  PMID: 22686199
Inclusion Body Myopathy with Paget Disease of Bone and Frontotemporal Dementia (IBMPFD); Valosin-containing protein (VCP); global microarray; signaling intermediates and cascades; actin cytoskeleton; autophagy; lysosome; growth factors; FoxO transcription factor
8.  Differential gene expression and a functional analysis of PCB-exposed children: Understanding disease and disorder development 
Environment International  2011;40:143-154.
The goal of the present study is to understand the probable molecular mechanism of toxicities and the associated pathways related to observed pathophysiology in high PCB-exposed populations. We have performed a microarray-based differential gene expression analysis of children (mean age 46.1 months) of Central European descent from Slovak Republic in a well-defined study cohort. The subset of children having high blood PCB concentrations (>75 percentile) were compared against their low PCB counterparts (<25 percentile), with mean lipid-adjusted PCB values of 3.02±1.3 and 0.06±0.03 ng/mg of serum lipid, for the two groups, respectively (18.1±4.4 and 0.3±0.1 ng/ml of serum). The microarray was conducted with the total RNA from the peripheral blood mononuclear cells of the children using an Affymetrix platform (GeneChip Human genome U133 Plus 2.0 Array) and was analyzed by Gene Spring (GX 10.0). A highly significant set of 162 differentially expressed genes between high and low PCB groups (p value <0.00001) were identified and subsequently analyzed using the Ingenuity Pathway Analysis tool. The results indicate that Cell-To-Cell Signaling and Interaction, Cellular Movement, Cell Signaling, Molecular Transport, and Vitamin and Mineral Metabolism were the major molecular and cellular functions associated with the differentially altered gene set in high PCB-exposed children. The differential gene expressions appeared to play a pivotal role in the development of probable diseases and disorders, including cardiovascular disease and cancer, in the PCB-exposed population. The analyses also pointed out possible organ-specific effects, e.g., cardiotoxicity, hepatotoxicity and nephrotoxicity, in high PCB-exposed subjects. A few notable genes, such as BCL2, PON1, and ITGB1, were significantly altered in our study, and the related pathway analysis explained their plausible involvement in the respective disease processes, as mentioned. Our results provided insight into understanding the associated molecular mechanisms of complex gene-environment interactions in a PCB-exposed population. Future endeavors of supervised genotyping of pathway-specific molecular epidemiological studies and population biomarker validations are already underway to reveal individual risk factors in these PCB-exposed populations.
doi:10.1016/j.envint.2011.07.008
PMCID: PMC3247643  PMID: 21855147
PCB; Microarray; Gene expression; Environmental exposure; Functional analysis; PCB-exposed population
9.  Analysis of the toxicogenomic effects of exposure to persistent organic pollutants (POPs) in Slovakian girls: correlations between gene expression and disease risk 
Environment International  2011;39(1):188-199.
The chemical composition of Persistent Organic Pollutants (POPs) in the environment is not uniform throughout the world, and these contaminants contain many structurally different lipophilic compounds. In a well-defined study cohort in the Slovak Republic, the POP chemicals present in the peripheral blood of exposed children were chemically analyzed. The chemical analysis data revealed that the relative concentration and profile of structurally different organic pollutants, including polychlorinated biphenyls (PCBs), 2,2’-bis(4-chlorophenyl)-1,1- dichloroethylene (p,p’-DDE), 2,2’-bis(4-chlorophenyl)-1,1,1-trichloro-ethane (p,p’-DDT), hexachlorobenzene (HCB) and β-hexachlorocyclohexane (β-HCH), may vary from individual to individual, even within the same exposure area. These chemicals can be broadly classified into two groups. The first group, the PCB congeners, primarily originated from industrial compounds and their byproducts. The second group of compounds originated from or was commonly used in the agricultural sector (e.g., DDT, HCB). The objective of this study was to examine the effects of the two POP exposure profiles on gene expression. For the study population, we selected prepubertal girls (mean age of 46.2 ± 1.4 months) with high POP concentrations in their blood (> 75% tile of total POP) and classified them in the high ‘PCB’ group when the total PCB concentration was significantly higher than the total concentration of other POP components and in the ‘Other Than PCB’ (OTP) group, when the total PCB concentration was significantly lower than the concentration of the other major POP constituents. A matched control group of girls (< 25% tile of total POP) was selected for comparison purpose (n = 5 per group). Our aims were to determine whether there were any common effects of high POP exposure at a toxicogenomic level and to investigate how exposure may affect physiological functions of the children in two different exposure scenarios. Global gene expression analysis using a microarray (Affymetrix Gene Chip Human genome U133 Plus 2.0 Array) platform was conducted on the total RNA of peripheral blood mononuclear cells from the girls. The results were analyzed by Partek GS, Louis, MI, which identified twelve genes (ATAD2B, BIVM, CD96, CXorf39, CYTH1 ETNK1, FAM13A, HIRA, INO80B, ODG1, RAD23B, and TSGA14) and two unidentified probe sets, as regulated differentially in both the PCB and OTP groups against the control group. The qRT-PCR method was used to validate the microarray results. The Ingenuity Pathway Analysis (IPA) software package identified the possible molecular impairments and disease risks associated with each gene set. Connective tissue disorders, genetic disorders, skeletal muscular disorders and neurological diseases were associated with the 12 common genes. The data therefore identified the potential molecular effects of POP exposure on a genomic level. This report underscores the importance of further study to validate the results in a random population and to evaluate the use of the identified genes as biomarkers for POP exposure.
doi:10.1016/j.envint.2011.09.003
PMCID: PMC3259908  PMID: 22208759
Persistent Organic Pollutants; POP; Gene Environment Interaction; Gene Expression; Human PBMC; IPA Analysis; Disease and Disorders
10.  The role of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) in mediating autophagy in myositis skeletal muscle: A potential non-immune mechanism of muscle damage 
Arthritis and rheumatism  2011;63(11):3448-3457.
Objective
Multinucleated cells are relatively resistant to classical apoptosis, and the factors initiating cell-death and damage in myositis are not well defined. We hypothesized that non-immune autophagic cell death may play a role in muscle fiber damage. Recent literature indicates that tumor necrosis factor-alpha-related apoptosis inducing ligand (TRAIL) may induce both NFκB (nuclear factor kappa-light chain enhancer of activated B cells) activation and autophagic cell death in other systems. Here, we have investigated its role in cell death and pathogenesis in vitro and in vivo using myositis (human and mouse) muscle tissues.
Methods
Gene expression profiling indicated that expression of TRAIL and several autophagy markers was specifically upregulated in myositis muscle tissue; these results were confirmed by immunohistochemistry and immunoblotting. We also analyzed TRAIL-induced cell death (apoptosis and autophagy) and NFκB activation in vitro in cultured cells.
Results
TRAIL was expressed predominantly in muscle fibers of myositis, but not in biopsies from normal or other dystrophic-diseased muscle. Autophagy markers were upregulated in human and mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NFκB activation and IκB degradation in cultured cells that are resistant to TRAIL-induced apoptosis but undergo autophagic cell death.
Conclusion
Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NFκB and autophagic cell death in myositis. Thus, this non-immune pathway may be an attractive target for therapeutic intervention in myositis.
doi:10.1002/art.30530
PMCID: PMC3203318  PMID: 21769834
11.  Glandular Gene Expression of Sinus Mucosa in Chronic Rhinosinusitis with and without Cystic Fibrosis 
Secretory cells in submucosal glands (SMGs) secrete antibacterial proteins and mucin glycoproteins into the apical lumen of the respiratory tract, and these are critical for innate immune mucosal integrity. Glandular hyperplasia is manifested in diseases with obstructive respiratory pathologies associated with mucous hypersecretion, and is predominant in the sinus mucosa of patients with chronic rhinosinusitis (CRS), cystic fibrosis (CF), and clinical symptoms of CRS. To gain insights into the molecular basis of SMG hyperplasia in CRS, gene expression microarray analyses were performed to identify the differences in global and specific gene expression in the sinus mucosa of control, CRS, and CRS/CF patients. A marked up-regulation of 11 glandular-associated genes in CRS and CRS/CF sinus mucosa was evident. The RNA and protein expressions of the four most highly up-regulated genes (DSG3, KRT14, PTHLH, and OTX2) were evaluated. An increased expression of DSG3, KRT14, and PTHLH was demonstrated at the mRNA and protein levels in both CRS and CRS/CF sinus mucosa, whereas the increased expression of OTX2 was evident only for CRS/CF sinus mucosa, implicating OTX2 as a CF-specific gene. Immunofluorescence analysis localized DSG3, PTHLH, and OTX2 to serous cells, and KRT14 to myoepithelial cells, in SMGs. Because glandular hyperplasia is a central histologic feature of CRS, the identification of overexpressed glandular genes in the sinus mucosa lays the groundwork for future studies of glandular hyperplasia, and may ultimately lead to the development of novel treatments for mucous hypersecretion in patients with CRS.
doi:10.1165/rcmb.2010-0133OC
PMCID: PMC3175585  PMID: 21177983
chronic rhinosinusitis; cystic fibrosis; submucosal glands; hyperplasia; microarrays
12.  Metabolic remodeling agents show beneficial effects in the dystrophin-deficient mdx mouse model 
Skeletal Muscle  2012;2:16.
Background
Duchenne muscular dystrophy is a genetic disease involving a severe muscle wasting that is characterized by cycles of muscle degeneration/regeneration and culminates in early death in affected boys. Mitochondria are presumed to be involved in the regulation of myoblast proliferation/differentiation; enhancing mitochondrial activity with exercise mimetics (AMPK and PPAR-delta agonists) increases muscle function and inhibits muscle wasting in healthy mice. We therefore asked whether metabolic remodeling agents that increase mitochondrial activity would improve muscle function in mdx mice.
Methods
Twelve-week-old mdx mice were treated with two different metabolic remodeling agents (GW501516 and AICAR), separately or in combination, for 4 weeks. Extensive systematic behavioral, functional, histological, biochemical, and molecular tests were conducted to assess the drug(s)' effects.
Results
We found a gain in body and muscle weight in all treated mice. Histologic examination showed a decrease in muscle inflammation and in the number of fibers with central nuclei and an increase in fibers with peripheral nuclei, with significantly fewer activated satellite cells and regenerating fibers. Together with an inhibition of FoXO1 signaling, these results indicated that the treatments reduced ongoing muscle damage.
Conclusions
The three treatments produced significant improvements in disease phenotype, including an increase in overall behavioral activity and significant gains in forelimb and hind limb strength. Our findings suggest that triggering mitochondrial activity with exercise mimetics improves muscle function in dystrophin-deficient mdx mice.
doi:10.1186/2044-5040-2-16
PMCID: PMC3482394  PMID: 22908954
Duchenne muscular dystrophy; Muscle; AICAR; GW501516; Metabolism
13.  Global gene expression and Ingenuity biological functions analysis on PCB 153 and 138 induced human PBMC in vitro reveals differential mode(s) of action in developing toxicities 
Environment international  2011;37(5):838-857.
Several reports have indicated that low level of polychlorinated biphenyl (PCB) exposure can adversely affect a multitude of physiological disorders and diseases in in vitro, in vivo, and as reported in epidemiological studies. This investigation is focused on the possible contribution of two most prevalent PCB congeners in vitro in developing toxicities. We used PCB 138 and 153 at the human equivalence level as model agents to test their specificity. We chose a global approach using oligonucleotide microarray technology to investigate modulated gene expression for biological effects, upon exposure of PCBs, followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We performed in vitro studies with human peripheral blood mononuclear cells (PBMC), where PBMC cells were exposed to respective PCBs for 48 hrs. Overall, our observation on gene expression indicated that PCB produces a unique signature affecting different pathways, specific for each congener. While analyzing these data through IPA, the prominent and interesting disease and disorders were Neurological disease, Cancer, Cardiovascular disease, respiratory disease, as well as endocrine system disorders Genetic disorders, and reproductive system disease. They showed strong resemblances with in vitro, in vivo, and in the epidemiological studies. A distinct difference was observed in renal and urological diseases, organisimal injury and abnormalities, dental disease, ophthalmic disease, and psychological disorders, which are only revealed by PCB 138 exposure, but not in PCB 153. The present study emphasizes the challenges of global gene expression in vitro and was correlated with the results of exposed human population. The microarray results give a molecular mechanistic insight and functional effects, following PCB exposure. The extent of changes in genes related to several possible mode(s) of action highlights the changes in cellular functions and signaling pathways that play major roles. In addition to understanding the pathways related to mode of action for chemicals, these data could lead to the identification of genomic signatures that could be used for screening of chemicals for their potential to cause disease and developmental disorders.
doi:10.1016/j.envint.2011.02.010
PMCID: PMC3097535  PMID: 21470681
PCB 138; PCB 153; Human PBMC; Gene expression; IPA Analysis; Disease and Disorders
14.  Sepsis Alters the Megakaryocyte–Platelet Transcriptional Axis Resulting in Granzyme B–mediated Lymphotoxicity 
Rationale: Sepsis-related mortality results in part from immunodeficiency secondary to profound lymphoid apoptosis. The biological mechanisms responsible are not understood.
Objectives: Because recent evidence shows that platelets are involved in microvascular inflammation and that they accumulate in lymphoid microvasculature in sepsis, we hypothesized a direct role for platelets in sepsis-related lymphoid apoptosis.
Methods: We studied megakaryocytes and platelets from a murine-induced sepsis model, with validation in septic children, which showed induction of the cytotoxic serine protease granzyme B.
Measurements and Main Results: Platelets from septic mice induced marked apoptosis of healthy splenocytes ex vivo. Platelets from septic granzyme B null (−/−) mice showed no lymphotoxicity.
Conclusions: Our findings establish a conceptual advance in sepsis: Septic megakaryocytes produce platelets with acutely altered mRNA profiles, and these platelets mediate lymphotoxicity via granzyme B. Given the contribution of lymphoid apoptosis to sepsis-related mortality, modulation of platelet granzyme B becomes an important new target for investigation and therapy.
doi:10.1164/rccm.200807-1085OC
PMCID: PMC2654976  PMID: 19136373
blood platelets; sepsis; granzyme B; apoptosis
15.  Expression Profiling of Inflammatory Mediators in Pediatric Sinus Mucosa 
Objective
To evaluate gene expression by microarray analyses of inflammatory mediators in the sinus mucosa of children with or without chronic rhinosinusitis (CRS).
Design
Prospective molecular genetics analysis of sinus mucosa from pediatric CRS and control patients.
Subjects
Eleven CRS patients who underwent endoscopic sinus surgery and ten children who underwent craniofacial resection or neurosurgical procedures.
Interventions
Gene expression levels of sinus tissue from 6 CRS and 6 control patients were analyzed on Affymetrix HGU133 plus 2.0 array chips. mRNA expression levels of upregulated inflammatory/immune response genes, as well as cytokines of interest, were further evaluated by quantitative RT-PCR.
Results
Gene expression using the Plier algorithm yielded the most consistent grouping of samples. 96 genes were significantly up-regulated more than 2 fold, and 123 genes were down-regulated by at least 50% in the CRS sinus tissues compared with controls (p<0.05). GeneSpring analysis demonstrated significant changes in several ontology categories in the CRS samples, including inflammatory/immune response genes. The chemokines CXCL13 and CXCL5, serum amyloid A, serpinB4 and defensin B1 were highly upregulated (≥ 5-fold). Increased expression of these genes was validated by quantitative RT-PCR in an independent set of tissues. Expression levels of the cytokines IL5, IL6 and IL8 were similar in both cohorts; these results were validated by RT-PCR.
Conclusions
Microarray analyses of sinus mucosa in children with CRS showed an increased expression of inflammatory genes involved in innate and adaptive immune systems. This technology can be successfully used to identify genes implicated in the pathogenesis of pediatric CRS.
doi:10.1001/archoto.2008.505
PMCID: PMC2696810  PMID: 19153309
16.  Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle After Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades 
Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
PMCID: PMC2733097  PMID: 19787081
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance
17.  NKG2A AND CD56 ARE CO-EXPRESSED ON ACTIVATED TH2 BUT NOT TH1 LYMPHOCYTES 
Human immunology  2006;66(12):1223-1234.
NKG2A is commonly expressed on cytotoxic cells but has been found on activated T-Helper (TH) cells. In identifying novel differentiating markers between TH1 and TH2 lymphocytes, we focused on NKG2A expression. TH1 and TH2 cells were negatively isolated from healthy volunteers for microarray analysis and RT-PCR. Flow cytometry of quiescent and activated TH1 and TH2 cells was performed. Isolates were >95% pure CD3+CD4+ cells (TH1=90.3% and TH2=84.1%). Microarrays showed differential expression of NKG2A and NKG2C isoforms between TH1 and TH2 cells. RT-PCR showed greater expression of NKG2A in TH2 cells (4-fold) and NKG2C in TH1 cells (3-fold). Flow studies showed tripling of TH2 NKG2A with activation to 10.76±4.01% (p=0.05), a 23-fold increase in CD56 to 35±14.54% (p=0.03), and an increase in NKG2A+CD56+ double-positive cells to 3.04±1.38% (p=0.04). TH1 lymphocytes did not differ with activation. We identified co-induction of NKG2A and CD56 upon activation of TH2 cells. These cells would likely bind more HLA-E and show increased effector inhibition. Given that certain viruses are known to decrease MHC class I and thus HLA-E production by antigen presenting cells, activated TH2 cells would bind less HLA-E in this scenario. This would likely result in less effector inhibition and a relatively robust TH2 response.
doi:10.1016/j.humimm.2006.02.005
PMCID: PMC1851905  PMID: 16690409
TH1 Cells; TH2 Cells; Natural Killer Cells; CD56; Lymphocyte Activation; NK — Natural Killer Cell; HLA — Human Leukocyte Antigen; TH — T-Helper Cell; IL — Interleukin; IFN — Interferon; TCR — T Cell Receptor; PCR — Polymerase Chain Reaction; MHC — Major Histocompatibility Complex; PBMC — Peripheral Blood Mononuclear Cell

Results 1-17 (17)